The Interesting of Antifungal Effects of Novel In Vitro Fabrics of Stabilized ZnO Nanofluids

18 Pages·2016·2.01 MB·English

Advances in Nanoparticles, 2016, 5, 206-223
http://www.scirp.org/journal/anp
ISSN Online: 2169-0529
ISSN Print: 2169-0510

The Interesting of Antifungal Effects of
Novel In Vitro Fabrics of Stabilized ZnO Nanofluids
Fatemeh Katouzian1, Zahra Fakhroueian2*, Soheila Moradi Bidhendi3
1Department of Microbiology, Faculty of Advanced Sciences and Technology, Pharmaceutical Sciences (IAUPS), Tehran, Iran
2School of Chemical Engineering, College of Engineering, and IPE, University of Tehran, Tehran, Iran
3Microbiology Department, Razi Vaccine and Serum Research Institute, Karaj, Iran

How to cite this paper: Katouzian, F., Abstract
Fakhroueian, Z. and Bidhendi, S.M. (2016)
The Interesting of Antifungal Effects of No- According to the extent of fungal infections, to be chronic these such diseases and
vel In Vitro Fabrics of Stabilized ZnO Nan- recently the emerging issue of increased antibiotic resistance in fungal infections,
ofluids. Advances in Nanoparticles, 5, 206- most of scientists are going to find a proper way to replace antibacterial agent by sig-
223.
nificant semiconductor ZnO nanoparticles (NPs). They are well known to be one of
http://dx.doi.org/10.4236/anp.2016.54022
the most important and special metal oxide nanoparticles in pharmaceutical against

Received: September 15, 2016 the most common fungi. ZnO nanoparticles were synthesized using sol-gel, hydro-
Accepted: November 12, 2016 thermal and functionalized surface methods and formulated in water solutions as
Published: November 15, 2016
nanofluids. XRD, FTIR and SEM techniques and UV-Vis absorbance spectroscopy

characterized their ZnO modified nanostructures. Also antimycotic potential ac-
Copyright © 2016 by authors and
Scientific Research Publishing Inc. cording to generally tests such as: (MIC) minimum inhibitory concentration, (MFC)
This work is licensed under the Creative minimum fungicidal concentration and normally well diffusion method with stan-
Commons Attribution International dard strains fungi were performed. Among five common fungi strains using in this
License (CC BY 4.0).
research, new various ZnO nanofluids showed noticeable results for dermatophyte
http://creativecommons.org/licenses/by/4.0/
fungi like Trichophyton mentagrophytes, Microsporum gypseum, Microsporum ca-
Open Access
nis, Candida albicans and Candid tropicalis which had un growth zones in order 70,
40, 35, 30 and 30 mm in comparing with Clotrimazole reference reagent: 30, 25, 25,
18 and 20 mm by well method. The performance of MIC for ZnO nanofluids on fun-
gi was determined to be equal to 0.35, 3.12, 6.25, 6.25 and 6.25 µgr/ml and MFC of
nanoproducts showed the 1.5, 12.5, 25, 25 and 25 µgr/ml. Therefore, the designed
ZnO nanofluids could reveal the most effect on fungi which cause dermal (ring-
worm), mucosal (thrush) and vaginal infections, so we are able to apply these surface
high energetic ZnO water-based nanofluid formulations as in vitro nanomedicine
and nanohygiene for the first time.

Keywords
ZnO Nanofluids, Potential Medical Applications, Antifungal Activity,
DOI: 10.4236/anp.2016.54022 November 15, 2016
F. Katouzian et al.

Traditional Resistant Antibiotic, Surface Modification, Nanomedicine,
Nanohygiene, MFC, Nanoformulation, Stabilized Water-Based Nanoparticle

1. Introduction
Infections caused by fungi has a wide range, depending on the location were classified
to superficial cutaneous, mucosal, subcutaneous and systemic infection. Although sys-
temic infections can be fetal, but the level of improvement cutaneous is often limited
and curable. Fungi classified into two groups pathogenic and opportunistic. The first
group affects healthy people and second group affects susceptible individuals [1]. Re-
cently, serious searches for new antimicrobials have performed due to the appearance
of various infectious morbidity and improvement in occurrence of drug resistance
against some of pathogenic fungi and zinc oxide nanoparticles are the most promising
and novel therapeutic agents for this event. It was found that, the upsurge in the recent
research about the nanoparticles and interesting advantage application as antimicro-
bials are due to having individual physiochemical properties and biological toxicity
against growth inhibitory of microbes and fungi [2]. Main difference parameters cause
nanoparticles including 1 - 100 nm in diameter to show significantly chemical reactivity
differently than their bulk structures, and their properties are able to change with the
decrease in dimensions [3]. The surface of nanoparticles can be manipulated exclusive-
ly by physico-chemical, and optical reactions suitably for desired applications especially
in the field of untifungi and nanomedicine [4]. Nanoparticles have unique large surface
area, high surface-to-volume ratio, and energetic management atoms with small size
dependent chemical and physical properties that have dimensions comparable to bio-
logical functional units containing exceptional shape and morphology, so they are be-
ing explored extensively currently. These characteristic make them as a fascination
candidate for various application in both in vivo and in vitro antibacterial and antifun-
gal in the field of nanomedicine researches. ZnO nanoparticles (NPs) and significant
their nanofluids are using mainly in targeted drug delivery, imaging, sensing, antimi-
crobials to target highly pathogenic and great drug resistant microbes, cancer treatment
and artificial implants in modern medicine science. Biocompatibility is an extensive
potential for nanobiomedical application researches and most of the nanomaterials es-
pecially ZnO nanoparticles indicate this trait [5]. The antifungal applications of two
nanometal oxide ZnO and MgO nanoparticles (NPs) with nearly average size of 30 and
50 nm, respectively are considered on some fungal infections [6]. R.V. Ravishankar et
al. [2] also investigated that ZnO and CuO (NPs) owing to highly antiseptic and fungi-
cides nature are being combined towards a diversity of pharmaceutical, medicinal sub-
stance and skin coatings. Recently, the scientists are going to find a proper way to re-
place old and traditional nanomedicine by novel materials such as effective ZnO nano-
particles and special nanofluids as new drugs, due the extent of fungal infections and
increasing antibiotic resistant and lack of new control strategies [7]. ZnO NPs and their
207
F. Katouzian et al.

novel water based nanofluids showed that could be potent pharmaceutical against the
most common fungi, with high strong control the growth of fungi. One of the impor-
tant and usual applications of ZnO nanoparticles is wallpapers in hospitals similar to
fungicide and antimicrobials. Because the strong antifungal effect against bacterial pa-
thogens and specific fungi, ZnO (NPs) is also an effective component in producing of
dermatophytic lesions to form of antifungal creams, pomades, and lotions [8]. It was
reported that superficial fungal infections (SFI) are prevalent skin diseases and affect
millions of people worldwide [9]. Earlier most dermatophyte strains had relatively re-
stricted geographical distribution but currently, dermatophytosis has become one of the
most common human infectious diseases worldwide. It is obvious that, the environ-
mental bacterial and fungal pollution contributes a major problem to human and ani-
mal health. Dermatophytes are fungi that require keratin for growth. These fungi can
cause superficial infections of the skin, hair, and nails. Dermatophytoses are referred to
as “tinea” infections. They are also named for the body site involved. Microsporum,
Trichophyton, and Epider-mophyton species are the well-known pathogens in skin in-
fections. Ring worm, athlete’s foot, jock itch are also the main diseases cause by derma-
tophytes fungi [10]. Also, Candida albicans and Candida tropicalis are in group of no-
socomial candidemia. Candidiasis is usually a very localized infection of the skin or
mucosal membranes, including the oral cavity (thrush), the pharynx or esophagus, the
gastrointestinal tract, the rectum, anus, perianal/perirectal or anorectal area (in men as
well as women), the perineum, the urinary bladder, the fingernails or toenails (ony-
chomycosis), and the genitalia (vagina, penis, etc.) [11] [12]. The patient people who
have HIV/AIDS and lost their immune system can increase oral candidiasis infection
more than healthy ones, especially for adult people with wearing dentures [13].
The present study was designed to conduct laboratory to evaluate in vitro inhibitory
effect of ten various stabilized formulations of water-based zinc oxide nanofluids on the
growth of five dermatophyte and systemic fungi for the first time and all the results
were compared with clotrimazole as an antifungal medicine standard reagent. It is cer-
tainly, we can apply them in nanomedical soon, because these manufacturing are very
novel technology in medicine industry.
2. Materials and Experimental Details
2.1. Synthesis and Formulation of New Soluble ZnO Nanofluids
ZnO nanoparticles (NPs) were prepared by chemical hydrolysis as sol-gel process,
wet-chemical procedures, and followed by hydrothermal method [14]. The surface of
synthezied ZnO NPs were functionalized to hydrophilic and modified by specific rea-
gents, and carried out in aqueous media and formulated using various nonionic surfac-
tants including different HLB, ethylene glycol solvent (EG), polyvinyl pyrrolidone
(PVP), oily herbal fatty acids, organosilane, water soluble solvents and suitable pH rea-
gents. We could synthesis of 10 novel nanoformulations in this study containing syn-
thezied ZnO NPs, and experimented through 5 various fungal strains.
208
F. Katouzian et al.

2.2. Introducing of 10 Novel Various ZnO Nanoformulations with
Their Numeric Codes
Sample 1—with ZnO 48 code, the synthezied ZnO NPs powder was soluble in mix-
ture of water and alcohol, and dimethyldichloro silane in EG solvent which is amphi-
philic and soluble in water.
Sample 2—with ZnO 51 code, ZnO NPs powder modified by Soya fatty acid was
dissolved in water and 1ml organosilane, and pH was adjusted by citric acid.
Sample 3—with ZnO 391code, ZnO NPs powder was functionalized with multiwall
CNT (carbon nano tubes), then dissolved in mixture of distilled water, ethylbanzene
solvent, and LA2EO (lauryl alcohol 2-EO) as nonionic surfactant.
Sample 4—with ZnO 45 code, ZnO NPs powder was capped with green Soya fatty
acid, then dissolved in nano PVP polymer and PEG 4000 mixed in liquid distilled water.
Sample 5—Synthesis of ZnO/PVP nanocomposites, 0.008 mole zinc acetate dihydrate
was hydrolyzed by 0.09 mole liquid KOH. Then the 2 - 4 gram PVP polymer was added
and refluxed during 6 - 8 h. The supernatant product was evaporated and washed with
mixture of water and alcohol solution, then it was dried for 6 hours at 80˚C in oven to
obtain the precursor.
Sample 6—with ZnO 175C code, 0.01 gr ZnO NPs powder modified with CNT was
dissolved in mixture of LA2EO as emulsifier for water-oil emulsion, cyclohexane and
vinyl acetate solvents. Then ZnO/PVP nanocomposites containing alcohol was added.
Sample 7—with ZnO 95 code, in this nano formulation, 0.02 gr ZnO NPs powder
was added to 2 ml Triton-X-100 and 3 ml LA3-EO (lauryl alcohol 3-EO) in 2-butanol
alcohol. Hydroxy propyl cellulose as stabilizer containing CaO NPs in liquid water were
added and mixed completely.
Sample 8—with ZnO 175P code, this nanoproduct is similar to the sample of No.6,
but we used a mixture of 2 ml Span 80 as emulsifier, solubilizer and stabilizer in 1 ml
organosilane as dispersing agents in liquid water.
Sample 9—with ZnO 109 P3 code, it was fabricated from ZnO and TiO NPs along
2
with a small amount of sodium dodecyl sulfate (SDS) as anionic surfactant and PEG
4000 in alcohol solvent contains acidic pH value.
Sample 10—with ZnO 176D code, the mixture of zinc nitrate and zinc chloride were
hydrolyzed with HO solution included polyvinyl alcohol (PVA) stabilizer and LA3EO
2 2
nonionic hydrophilic surfactant. The obtained black product was washed several times
and annealed at 400˚C for 6 hr. 0.05 gr ZnO NPs product was dissolved in ethylbanzene
and vinyl acetate solvents contained acidic pH value through liquid water along with 1
ml Span 80. This nanoformulation could mix strongly in order to obtain homogeneous
ZnO water solution nanofluid.
2.3. Fungi Strains and Culture Media, and Typical Bacteriological
Tests for Considering of Ten New Water-Based
ZnO Nanofluids with Dermatophytes and Candida Spp.
Table 1 and Table 2 illustrate the significant biological reactions against several fungi
209
F. Katouzian et al.

Table 1. Evaluation of ten ZnO nanofluids and nanoformulations as antifungal drugs against Candida species activity.
Well method (mm)

CL
ZnO48 ZnO51 ZnO391 ZnO45 ZnO/PVP ZnO175C ZnO95 ZnO175P ZnO109P3 ZnO176D
MIC (µgr/ml) 300 µgr/ml
MFC (µgr/ml)
Microbial strains
18 30 20 20 25 - - 25 - 20
Candida
albicans 50 6.25 25 25 12.5 - - 12.5 - 25 18 mm
PTCC5027
100 25 100 100 50 - - 50 - 100
- 20 20 15 15 15 15 30 20 20
Candida
tropicalis - 25 25 50 50 50 50 6.25 25 25 20 mm
PTCC5028
- 100 100 100 100 100 100 25 100 100

Table 2. Evaluation of ten ZnO nanofluids as antifungal drugs against typical dermatophytes species activity.
Well method (mm)

CL
MIC (µgr/ml) ZnO48 ZnO51 ZnO391 ZnO45 ZnO/PVP ZnO175c ZnO95 ZnO175P ZnO109P3 ZnO176D
300 µgr/ml
MFC (µgr/ml)
Microbial strains
25 25 - 35 30 30 20 20 25 30
Microsporum canis
12.5 12.5 - 6.25 6.25 6.25 25 25 12.5 6.25 25 mm
PTCC5069
50 50 - 25 25 25 100 100 50 25
20 30 50 40 30 70 40 40 30 30
Trichophyton
mentagrophytes 25 6.25 1.56 3.12 6.25 0.75 3.12 3.12 6.25 6.25 30 mm
PTCC5054
100 25 6.25 12.5 25 3.12 12.5 12.5 25 25
35 20 20 28 - 35 - 40 15 10
Microsporum
gypseum 6.25 25 25 6.25 - 6.25 - 3.12 50 100 25 mm
PTCC5070
50 100 100 25 - 25 - 12.5 100 100

related to Clotrimazole (300 µg/ml) as standard reference by well diffusion, MFC, and
MIC methods. Therefore, for evaluation of antifungal activity, five strains obtained
from the Persian Type Culture Collection (PTCC), Tehran, Iran, including Microspo-
rum gypseum (PTCC 5070), Microsporum canis (PTCC 5069), Trichophyton menta-
grophytes (PTCC 5054), Candida albicans (PTCC5027) and Candida tropicalis
(PTCC5028) were provided. All culture were applied in this project such as: Sabouraud
dextrose agar (SDA, Merck), Sabouraud dextrose broth (SDB, Merck), Dermatophyte
selective agar (DTM agar, Merck).
2.3.1. Well Diffusion Method
All fungi were cultured in medium for fresh culture, after time growth, then starting to
do antimicrobial methods. ZnO nanofluids were tested in vitro for their antifungal ac-
tivities against strains by the agar diffusion technique [14]. The tested samples were
dissolved in water to prepare chemicals of stock solutions. The suspension of patho-
genic fungi were cultured on Sabouraud dextrose agar and DTM agar media (Merck),
210
F. Katouzian et al.

respectively in Petri dishes with an inner diameter 9 cm to provide thin agar plates after
solidification of thickness 3.4 - 3.5 mm. After solidification, hollows of 5 mm diameter
wells were cut from the agar using a sterile cork-borer, and 0.1 ml of each of the tested
solutions were poured into the wells. The Petri dishes were incubated at 5˚C - 8˚C for
2 - 3 h to permit good diffusion and then incubated for 24 - 48 h for yeast and 5 - 7 days
for dermatophytes fungi at 28˚C. After incubation the diameter of inhibition zone
(mm) was measured. Clotrimazole were purchased from Padtan Teb Company and
used in a concentration of 300 μg/ml as standard antifungal references [15].
2.3.2. Determination of Minimum Inhibitory Concentration Method (MIC)
The aim of broth methods is to determine the lowest concentration of the assayed an-
timicrobial agent (minimal inhibitory concentration, MIC) that, under defined test
conditions, inhibits the visible growth of the fungi being investigated. MIC values are
used to determine susceptibilities to drugs and also to evaluate the activity of new anti-
microbial agents. For broth dilution, often determined in 13 tubes format, fungi are in-
oculated into a liquid growth medium in the presence of different concentrations of
ZnO nanofluid. Growth is assessed after incubation for a defined period of time for
Candida spp. (24 - 48 hrs.) and dermatophytes species (4 - 7 days) and then the last
tube of non-growth are MIC value [16].
2.3.3. Determination of Minimum Fungicidal Concentration Method (MFC)
Turbidity indicates growth of the microorganism and the MIC is the lowest concentra-
tion where no growth is visually observed. To determine the MFC, the dilution
representing the MIC and at least two of the more concentrated test product dilutions
are plated for 24 - 48 hrs for Candida species and 4 - 7 days for dermatophytes species,
then enumerated to determine viable CFU/ml. The MFC is the lowest concentration
that demonstrates a pre-determined reduction (such as 99.9%) in CFU/ml when com-
pared to the MIC dilution [17]. Table 1 show all of these very significant results for
Candida spp. and Table 2 indicate dermatophytes species activities.
2.3.4. The Presentation of Two Tables Due to Determination of Ten New
ZnO Nanofluids against Five Microbial Strain Fungal Pathogens
Table 1 illustrates the significant biological reactions against several fungi related to
Clotrimazole (300 µg/ml) as reference. For the evaluation of antifungal activity, the re-
sults showed that among of ten different nanoformulation of ZnO nanofluids, sample 2
(ZnO51), displays the best effect on C. albicans in well method with inhibition zone 30
mm, MIC 6.25 µgr/ml, and MFC 25 µgr/ml. So, the sample 8 (ZnO175P), illustrates the
best effect on C. trupicalis in well method with inhibition zone 30 mm, MIC 6.25 µgr/
ml, and MFC 25 µgr/ml in comparing with Clotimazole (18 and 20 mm as references )
respectively.
Table 2 exhibits that among of various ten nanoformulation of ZnO nanofluids,
sample 4 (ZnO45), demonstrates the best effect on M. canis in well method with inhibi-
tion zone 35 mm, MIC 6.25 µgr/ml, and MFC 25 µgr/ml. Sample 6 (ZnO175C), intro-
duced the best effect on T. mentagrophytes in well method by inhibition zone 70 mm,
211
F. Katouzian et al.

MIC 0.75 µgr/ml, and MFC 3.12 µgr/ml. The sample 8 (ZnO175P) registers the best ef-
fect on T. mentagrophytes and Microsporum gypseum in well method with inhibition
zone 40 mm, MIC 3.12 µgr/ml, and MFC 12.5 µgr/ml in comparing with control (25,
30, and 25 respectively).
3. Results and Discussion
3.1. Characterization of ZnO Nanofluids
3.1.1. SEM Pictures of Synthezied ZnO Nanoparticles (NPs) and
Some Their Nanofluids
SEM images of ZnO/PVP NPs in Figure 1, and various stable nanofluids 4 and 6 sam-
ple solutions containing different morphologies were showed in Figure 2 and Figure 3.
ZnO NPs as interesting fine nanosphericals were located among the green fatty acid
layers indicated in Figure 4.

Figure 1. SEM images of ZnO/PVP nanocomposites as spherical nanoparticles.

Figure 2. SEM images of sample 4 (ZnO 45) nanofluid, according to interesting nano cubic formation.
212
F. Katouzian et al.

Figure 3. SEM images of sample 6 (ZnO175C) indicate homogeneous morphology for multiwall carbon nanotubes ((MWNTs)) which are
coated on ZnO NPs (typical surface modification).

Figure 4. SEM images of ZnO NPs immersed into Soya fatty acid layers, and dispersed in sample 4 (ZnO45) nanofluid solution as fine
spherical forms.

Figure 5 reveals typical surface morphology and structure pictures for uniformly
spherical ZnO NPs in Sample 1-with ZnO 48 code, and Sample 7-with ZnO 95 code
nanofluids.
3.1.2. XRD Pattern of ZnO NPs and ZnO Nanofluids Solutions
Figure 6 illustrates XRD pattern analyses of ZnO nanoparticles powder (a), and ZnO
NPs dispersed in nanofluids (b, c), which the pattern of ZnO NPs is compatible with
JCPDS card number 36 - 1451, recording the crystallographic structure of the nanopar-
ticles as typical pure hexagonal phase with Wurtzite structure including space group
p63 mc.
3.1.3. FTIR Spectrum of Hexagonal Structure of ZnO NPs and
Surface Modified in Nanofluids
FTIR spectroscopy (Bruker Co. model Tensor 27) for ZnO NPs (a), ZnO NPs immersed
in green fatty acids (b), and ZnO NPs dispersed in water-based fluid (c) were deter-
mined in Figure 7. A sharp peak at 493.50 cm−1 can be assigned to the Zn-O stretching
bond in ZnO NPs (a) and two low frequency at 619 - 857 cm−1 can be attributed to hy-
dride Zn-H bending modes. FTIR spectroscopy (b) shows a very sharp peak at 1357.12
cm−1 for –COO groups and also water –OH stretching band (at 3303.31 cm−1) of car-
boxylic acid group derivative of Soya fatty acid during surface modification process of
213
F. Katouzian et al.

Figure 5. SEM images of typical ZnO NPs powder as homogeneous nanospherical.

Figure 6. XRD curves of ZnO NPs powder as nanospherical form (a) without any surface mod-
ification, and (b) is belong to the ZnO NPs coated with green fatty acids, and (c) can point to fine
ZnO NPs modified with multiwall carbon nanotubes.

Figure 7. FTIR spectrum for (a) ZnO NPs;(b) ZnO NPs immersed in green fatty acids and (c)
sample 6 (ZnO 175C), which ZnO NPs incorporated on multiwall CNT.
214
F. Katouzian et al.

ZnO NPs [14]. In sample 6 (ZnO 175C) (c) it was found that the broad band at 3300.79
cm−1 can be attributed to –OH stretching vibration modes and very low frequency at
720. 06 cm−1 and the other at 1465. 25 cm−1 is also for typical multiwalled carbon nano-
tubes (MWNTs). In addition, a group of peaks in the 1600 - 1741.24 cm−1 range consti-
tutes the G-band due to a symmetry carbon structure, and one peak around 1373.22
cm−1, which called the D-band, is assigned to the presence of disorder in graphitic ma-
terials were seen in figure (c) spectroscopy. The C＝O groups of pure PVP show a
prominent peak at 1663.25 cm−1 in FTIR spectrum which are able to have interaction
between PVP and metal nanoparticles.
3.1.4. UV-Vis Spectrophotometer of ZnO NPs as Powder
The absorption spectrum of ZnO nanostructures as powder and nanofluids appeared
around of 190 - 196 cm−1 with max curves in the area of low wavelengths due to the
many active sites of the surface defects, which are potential surface luminescence cen-
tres. UV-Vis emission peaks can show very fine particle sizes in nanofluids containing
high band gaps and various nanostructural defects. Figure 8 indicates different kinds of
UV-Vis spectroscopies for two ZnO nanoproducts.
3.2. Result of Antifungal Activities Derivative of Table 1 and Table 2
According to the results of tests and Table 1 and Table 2, on various fungi in well
method showed that samples 1, 2, 4, 6, 5, 3, 8, 9, 10, and 7 had the greatest effect on the
fungus Trichophyton mentagrophytes (Ringworm disease). The best and highest results
on other fungi such as Trichophyton mentagrophytes, M. gypseum, M. canis, C. albi-
cans, Candida tropicalis with inhibition control to clotrimazole 70, 40, 35, 30, 30 mm
were measured (for diseases such as: alopecia, cutaneous and mucosal infections). In
the MIC test results on fungi showed that nanofluids with codes 1, 2, 4, 6, 5, 3, 8, 9, and
10, the largest number of nanofluids zinc oxide on Trichophyton mentagrophytes indi-

ZnO28
2.5 ZnO175C
2.5
196nm
2
2 190n
on 1.5
sorpti 1 ption1.5
Ab 0.5 bsor 1
A
0.5
0
0 200 400 600 800 0
-0.5
wavelength (nm) 0 200 400 600 800
wavelength (nm)

(a) (b)
Figure 8. The UV-Vis (Perkin Elmer model lambda 35) absorption spectrum of ZnO nanopro-
ducts are shown of two ZnO NPs (a), and sample 6 (ZnO 175C) nanofluid (b). It can be detected
that the absorption edge appeared toward strong-sharp peaks at 196 and 190 nm in the ultravio-
lent region. The band gap energy will be actually more than the 3.37 eV in the bulk structure, and
UV exciton emission were also shifted to blue shifts compared with ZnO bulk.
215